Effect of fluctuations in temperature encountered during handling and shipment of human cryopreserved semen.

The effect of temperature fluctuations which cryopreserved spermatozoa may undergo during routine shipping and handling was evaluated in sperm frozen with two cryoprotectants. Sperm frozen in TEST-yolk buffer maintained motility better than those frozen in glycerol solution in all studies. Sperm motility was significantly compromised in samples stored more than one day in dry ice, regardless of the cryoprotectant, and more than two days in a liquid nitrogen shipping dewar if frozen in glycerol solution. Sperm motility was not compromised following exposure to room temperature for up to 3 min if TEST-yolk buffer was used as the cryoprotectant, but was compromised following 1 min exposure using glycerol cryoprotectant. This study describes the limits of non-ideal conditions that spermatozoa may undergo during shipping and handling, and demonstrates the effects of the cryoprotectant on those limits.

The binding of recombinant human relaxin to human spermatozoa.

Porcine relaxin has been reported to stimulate various human sperm functions. In this paper we report that human recombinant relaxin binds to human sperm with a high affinity (Kd = 6.5 x 10(-10)). The bound 125I-relaxin was not displaced by insulin, or human chorionic gonadotropin, however, it was displaced by unlabeled relaxin. In sperm function studies, recombinant human relaxin stimulated sperm motility, zona-free hamster egg penetration, and the acrosome reaction.

Ovulation induction with gonadotropins and intrauterine insemination compared with in vitro fertilization and no therapy: a prospective, nonrandomized, cohort study and meta-analysis.

OBJECTIVES: To determine whether one to four cycles of ovulation induction with hMG and IUI or one cycle of IVF results in the highest pregnancy rate and is least expensive and whether published pregnancy rates for one to four cycles of hMG and IUI results in a higher pregnancy rate than rates for one cycle of IVF, zygote intrafallopian transfer (ZIFT), or GIFT. DESIGN: Prospective, nonrandomized, cohort study. Patients were excluded who were infertile for < 18 months, had a significant male factor, had greater than mild endometriosis, or had bilateral nonpatency of the fallopian tubes. Cohort groups included 47 hMG and IUI patients (99 cycles), 19 IVF patients (19 cycles), and 21 patients (210 cycles) receiving no treatment. A meta-analysis on accumulated hMG and IUI data using similar entry criteria was also performed. Theoretical calculations were performed and stable fecundity assumed to compare with national data on IVF, ZIFT, and GIFT. SETTING: Fertility Center, Division of Reproductive Endocrinology, University of Utah, Salt Lake City, Utah. RESULTS: A course of therapy with one to four cycles of hMG and IUI was just as effective as one cycle of IVF in achieving pregnancy. No significant difference in pregnancy rates was found between one IVF cycle and one to four cycles of hMG and IUI in our population. In vitro fertilization was more expensive than four cycles of hMG and IUI. Both IVF and hMG and IUI were more effective than no therapy. Published data also suggest that four cycles of hMG and IUI theoretically result in higher pregnancy rates than one cycle of IVF, ZIFT, or GIFT. CONCLUSION: Cost-benefit analysis comparing hMG and IUI, IVF, and no therapy in infertility patients may favor a course of four cycles of hMG and IUI as the first line of therapy. Using meta-analysis and theoretical assumptions, the pregnancy rate for one cycle of hMG and IUI is inferior to IVF, GIFT, or ZIFT; two cycles are comparable to IVF or ZIFT and inferior to GIFT; three cycles are superior to IVF or ZIFT and comparable to GIFT; and four cycles are theoretically superior to all techniques.

Nicotine, but not cotinine, has a direct toxic effect on ovarian function in the immature gonadotropin-stimulated rat.

PMSG-primed and hCG-triggered rat ovaries were exposed to nicotine and its major metabolite, cotinine, using in vitro and in vivo experimental approaches. In vivo, a dose-dependent reduction in oocytes within the fallopian tube was noted in nicotine treated rats (6.25 ng/g animal weight, P < 0.001). Serum estradiol concentrations were also reduced in rats receiving nicotine (P < 0.04). There were no significant differences in weight gain. Cotinine had no effects. In vitro, nicotine also caused a dose-dependent reduction in oocytes in the collection chamber (P < 0.0001). Estradiol levels in nicotine-treated perfusions were reduced and reached statistical significance at 7 h (P < 0.003). The in vitro fertilization rate was reduced for nicotine-treated perfusions exposed to 1.43 pg/mL of nicotine (P < 0.001). Cotinine had no effect in vitro. We conclude that nicotine inhibits ovulation, estradiol production, and fertilization both in vivo and in vitro in rat models of ovulation. Cotinine did not affect these parameters. These effects of nicotine are notably independent of nicotine's known effect on the midcycle gonadotropin surge.

The incidence of antisperm antibodies in infertility patients with a history of cryptorchidism.

Infertility in patients with a history of cryptorchidism is usually the result of oligo-asthenospermia. In this study we analyzed the incidence of antisperm antibodies in infertility patients with a history of cryptorchidism, general infertility patients and donors of known fertility. Of the cryptorchid patients 66% tested positive for antisperm antibodies compared to 2.6% of the control group of infertile patients and 2.8% of the donors of known fertility. Sperm progressive motility was significantly (p < 0.05) decreased in the cryptorchid patients testing positive for antisperm antibodies compared to those testing negative for antisperm antibodies, and compared to both control groups. Of the patients treated for cryptorchidism by orchiopexy 52% were positive for antisperm antibodies, and the mean patient age at orchiopexy was significantly (p < 0.01) higher in the positive antibody group (14.2 +/- 1.2 years old) than the negative antibody group (8.6 +/- 0.8 years old). While decreased sperm concentration, motility and morphology are usually the primary causes of infertility in patients with a history of cryptorchidism, it appears that the presence of antisperm antibodies is also increased in these patients, which may contribute to reduced fertility.

A functional analysis and the potential clinical significance of 7 categories of sperm morphology.

Sperm morphology is a critical factor in the evaluation of infertile men. Patients often exhibit a morphology profile with consistent increases of specific abnormal categories of sperm morphology. We analyzed the functional ability of 7 categories of sperm morphology using assays that measure sperm viability longevity, deoxyribonucleic acid packing, acrosomal status, and the stimulation of the acrosome reaction with human cumulus/oocyte complexes and calcium ionophore A23187. Each morphology category was separately analyzed initially and at 3, 6, 12, 18 and 24 hours of in vitro culture. The data indicate that all categories of abnormal sperm morphology exhibit various functional debilities. However, tapered sperm most closely approached normal sperm function. All abnormal categories possessed subpopulations of sperm that functioned normally to the limits of these assays. These data indicate that increases in any of the categories of abnormal sperm morphology may lead to decreased fertility due to diminished functional ability indicated by the assays performed in this study.

N-glycosylation of forms of angiotensin converting enzyme from four mammalian species.

To help clarify bases for the molecular weight and surface charge heterogeneities of forms of somatic angiotensin converting enzyme (ACE), we examined for differences in N-glycosylation. ACE preparations purified from human, guinea pig, rat and rabbit tissues were found to be heterogeneous in terms of numbers of N-glycosylated sites (7-8 sites per molecule of ACE) and in types of structures of oligosaccharides used for glycosylation (complex versus high mannose oligosaccharide contents). Our findings, taken with reports of potential N-glycosylation sites and amino acid sequencing data, indicate that ACE forms can differ in terms of degrees of glycosylation, sites of glycosylation and structures of attached oligosaccharide units.

A comparison of guinea pig serum angiotensin converting enzyme with forms of angiotensin converting enzyme from human, rat and rabbit tissues.

Guinea pig serum angiotensin converting enzyme (ACE) activities exceed ACE activities of other mammalian sera by as much as two magnitudes. To examine the possibility that guinea pig ACE has a superior catalytic efficiency, we purified it to apparent homogeneity and compared it to highly purified forms of ACE from human seminal plasma, rat lungs and rabbit lungs. The first 24 amino acid residues of guinea pig and rat ACE forms were 96% identical with the sequence of human ACE. Second order rate constants (kcat/Km) for guinea pig, human and rabbit forms of ACE on reaction with benzoyl-Phe-Ala-Pro were identical (1.6E-09 M-1 min-1). Their dissociation constants on reaction with the ACE inhibitor RAC-X-65 were within a narrow range (10-16 pM). Thus, the high ACE activity of guinea pig serum is owing to high enzyme concentration and not to superior catalytic efficiency.

Role of the cumulus in the selection of morphologically normal sperm and induction of the acrosome reaction during human in vitro fertilization.

This study analyzed the role that the cumulus oophorus may play in the selection of morphologically normal sperm and the induction of the acrosome reaction. Using the triple stain technique, sperm morphology and acrosomal status were compared between sperm that penetrated the cumulus during in vitro fertilization and sperm from outside the cumulus. The mean percentage of morphologically normal sperm in the samples was 54 +/- 2.8 and increased (p < 0.05) to 67 +/- 2.6 within the cumulus. Tapered sperm were significantly decreased (p < 0.05) within the cumulus. The percentage of sperm undergoing the acrosome reaction significantly increased (p < 0.05) from 14.5 +/- 1.5 to 24.5 +/- 1.9 when incubated with a cumulus mass, and further increased to 49 +/- 3.3 when incubated with mature, expanded cumulus tissue containing an oocyte. These data indicate that human cumulus oophorus plays an active role in the selection of morphologically normal sperm, and influences the ability of the sperm to undergo the acrosome reaction.

Pregnancies following implantation of an artificial fallopian tube in rabbits.

The first cases of pregnancies obtained in vivo in animals after implantation of a Teflon artificial fallopian tube model are presented. The Teflon prosthesis was implanted into the uterine cornua of rabbits and fixed subcutaneously in the abdominal wall. Five to eight blastocysts obtained from donor rabbits were transferred through the tubing into each recipient animal's uterus 2 to 14 days after placement of the device. On posttransfer day 10, normally developed pregnancies were visible and confirmed histologically in 4 of 7 animals.

Immunosuppressive activity and alpha interferon concentrations in human embryo culture media as an index of potential for successful implantation.

OBJECTIVE: To evaluate potential correlations between establishment of pregnancy and immunosuppressive activity secreted by the preimplantation embryo. DESIGN: To evaluate immunosuppressive activity, supernatants from preimplantation embryos were assessed for their ability to inhibit lymphocyte proliferation. Additionally, alpha interferon concentrations were also measured in these supernatants. We compared these parameters from embryo culture supernatants of women who did and did not achieve pregnancy after in vitro fertilization (IVF). Immunosuppression was assessed using a lymphocyte proliferation assay with concanavalin A (Con A) and phytohemagglutinin (PHA) as mitogens. SETTING: In vitro fertilization program at the University of Utah Medical Center. PARTICIPANTS: Couples less than 40 years of age, with normal semen quality and bilateral tubal obstruction. RESULTS: Immunosuppression calculated using the stimulation index (mean +/- SEM) in pregnant and nonpregnant women, respectively, were: Con A: 43.9 +/- 3.9 versus 19.1 +/- 10.1, P less than 0.04. PHA: 23.6 +/- 5.6 versus 12.5 +/- 12.8, P less than 0.02. Alpha interferon levels (mean +/- SD) in pregnant and nonpregnant women were not significantly different: 23.98 +/- 9.6 U/mL versus 24.79 +/- 2.5 U/mL. CONCLUSIONS: We conclude that pre-embryos with the capacity for successful implantation secrete greater amounts of immunosuppressive factors than those destined not to implant, as measured by Con A and PHA lymphocyte proliferation assays. Refinement of assay techniques and identification of the substances involved could have significant impact on IVF programs.

An evaluation of various treatments to increase sperm penetration capacity for potential use in an in vitro fertilization program.

OBJECTIVE: To select in vitro fertilization (IVF) patients with a low sperm penetration assay (SPA) value and to determine if the penetration rate, fertilization rate, and the pregnancy rate (PR) can be improved in these patients by treating sperm with refrigeration, calcium ionophore A23187, prostaglandin E1, prostaglandin E2, or heparin. DESIGN: The study consists of three parts: identification of patients with poor SPA values, analysis of treatments to improve the SPA value, and evaluation of the treatments to improve fertilization and PRs. RESULTS: The data indicate that treatment of sperm with refrigeration can improve fertilization and PRs during IVF in selected patients previously shown to have an improved SPA value with the treatment. CONCLUSIONS: Three points are emphasized: (1) the treatments analyzed in this study can improve SPA values in some of the patients with low sperm penetration capacity; (2) of the treatments studied, sperm refrigeration resulted in the largest improvement in sperm penetration capacity; and (3) sperm refrigeration can increase fertilization and PRs during IVF in this select group of patients.

Ultrasonographic characteristics of the seminal vesicles after ejaculation.

A total of 12 men from a sperm donor bank underwent transrectal ultrasonography before and after ejaculation to investigate possible changes that could influence interpretation of seminal vesicle ultrasonography. The mean length of the seminal vesicles was 2.98 cm. before and 2.95 cm. after ejaculation. The anteroposterior diameter decreased only from 0.59 to 0.52 cm. Minimal differences were noted between the right and left seminal vesicles, and the greatest variation in size in an individual was only 3 mm. These results indicate that interpretation of seminal vesicle anatomy as seen by ultrasonography does not depend upon the temporal relationship to ejaculation.

Developing an artificial fallopian tube: successful in vitro trials in mice.

This report describes the design and testing of an artificial fallopian tube for the treatment of tubal infertility. Within the device, mouse eggs incubated with sperm were fertilized and a microinfusion pump was used to transport the fertilized ova through the tube. Normal offspring resulted from transfer of the developing embryos into pseudopregnant recipients. These results provide encouraging evidence that an artificial fallopian tube warrants further investigation as a potential alternative to in vitro fertilization.

A fifteen-year study of alterations in semen quality occurring after vasectomy reversal.

Semen quality was evaluated in 256 men at 2, 4, 6, and 12 months after vasectomy reversal. Total sperm counts were normal and averaged 91 X 10(6) sperm per ejaculate. The percentage of live sperm was decreased below normal to 36% and hypo-osmolarity values indicated only 41% of the sperm with normal membranes. Progressive motility and the total progressively motile sperm count were reduced and averaged 25% and 27 million, respectively. Approximately 50% of the patients had positive titers of sperm agglutinating and/or sperm immobilizing antibodies. Sperm morphology indicated tail and head shape defects with increases in both tapered and immature sperm. A fertility score combining several sperm parameters averaged only 37% of normal. All of these values remained constant with time after reversal except for progressive motility (increased), oval head shapes (increased), and tapered sperm (decreased). This study has demonstrated that characteristic defects occur in sperm (decreased). This study has demonstrated that characteristic defects occur in sperm motility, sperm membrane function and morphology, in addition to specimen fertility scores, after a vasectomy reversal and need to be taken into consideration when advising and treating these patients for subsequent fertility difficulties.

The gamete and embryo compatibility of various synthetic polymers.

Several popular and well-characterized polymeric materials were evaluated for their biocompatibility toward the cells unique to reproduction. To accomplish these studies, several in vitro tests were developed that evaluated biocompatibility between the polymers and spermatozoa, ova, and embryos. The data indicated significant differences between the materials with respect to their biocompatibility toward sperm motility, the sperm's ability to penetrate zona-free hamster eggs, and the ability of two-cell mouse embryos to divide. Polytetrafluoroethylene (PTFE-Teflon; PTFE, Chemplast Inc., Wayne, NJ), polyethylene glycol (PEG), and polyhydroxyethyl methacrylate (PHEMA) appear to be the most inert of the materials studied. Polyvinyl chloride (PVC; Tygon-Norton, Akron, OH) was found to be the most detrimental material toward gametes and embryos, with gross physiologic and morphologic changes observed in the PVC-exposed cells.

Artificial insemination: a comparison of pregnancy rates with intrauterine versus cervical insemination and washed sperm versus serum swim-up sperm preparations.

A four-cycle artificial insemination protocol was undertaken, with luteinizing hormone (LH) timing, to compare washed sperm and serum swim-up sperm preparations and cervical and intrauterine insemination. Of the 75 patients entered into the protocol series, 63 finished all 4 cycles. During the treatment period, 24 patients became pregnant, 20 of whom were donor (AID) pregnancies (out of 52 patients) and 4 were husband (AIH) pregnancies (out of 11 patients). Seventy-five percent of the pregnancies were intrauterine, whereas only 25% were from cervical inseminations. Sixty-two percent of the pregnancies were due to serum swim-up sperm preparations, and 38% were due to washed sperm preparations. The data indicate substantially higher pregnancy rates can be obtained with intrauterine insemination and suggest that additional studies are necessary to determine if the method of sperm preparation significantly influences pregnancy rates.

Ultrasound and spermatogenesis in the rat.

Using exposure conditions comparable to those which have been associated by others with positive results, we tested for effects of ultrasound on sperm production over a period of 12 weeks following treatment. Continuous wave exposure with spatial average intensities of 1, 2 and 4 W/cm2 and exposure times up to 10 minutes were used. In some experiments, the exposures were repeated after an elapsed time of 48 h. No significant changes in spermatogenesis were related to any of the exposure conditions in spite of the fact that some of the treatments caused thermal tissue damage near bone. No effects of exposure were found in weights of the testis, prostate, seminal vesicle, or whole body.

A 15-year followup of a nonmicrosurgical technique for vasovasostomy.

A 15-year followup was summarized for patients who had nonmicrosurgical vasectomy reversal. Over-all pregnancy rates were 45 per cent (43 per cent for the first 8 years and 49 per cent for the next 7 years) and 73 per cent of the pregnancies occurred within 1 year after vasectomy reversal. Patients who achieved pregnancy compared to those who did not had significantly better sperm motility and a lower incidence of antisperm antibodies. The data indicate that nonmicrosurgical techniques can be used successfully to reverse vasectomy more economically and more rapidly than the microsurgical approach, while contributing to a reasonable pregnancy rate.

PIP: A retrospective survey is presented of pregnancies achieved by 199 men who had nonmicrosurgical vasovasostomy between 1979 and 1986. Of the 73 who responded, 37 fathered children. The mean time between operation and pregnancy was 13 months, with 73% within 1 year, 13% within 2 years, 6% within 3 years, and 8% within 4 years or more. Semen analysis showed sperm at least once in 81%. Those who achieved pregnancy had significantly better sperm motility and lower incidence of antisperm antibodies, but no differences in sperm counts or sperm morphology. Of the 37 unsuccessful in producing pregnancies, 4 wives were subsequently diagnosed with infertility. Men with antisperm antibodies could sometimes be helped by giving them prednisone (20 mg daily) and performing artificial insemination with washed sperm during luteinizing hormone-treated cycles. The overall pregnancy rate reported here, 49%, is within the range recorded in 25 other published studies.

The Young-Dees operation for the correction of retrograde ejaculation.

Retrograde ejaculation can be a significant problem when it interferes with fertility. Sympathomimetic drugs and sperm retrieval from the urine with insemination of the wife are the common methods of treatment. When these methods have not been successful we have used the Young-Dees type of bladder neck reconstruction to convert retrograde ejaculation into normal or antegrade ejaculation. The operation has been successful in 4 of 5 patients.